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. Author manuscript; available in PMC: 2023 Jul 3.
Published in final edited form as: Nature. 2022 Oct 19;610(7933):775–782. doi: 10.1038/s41586-022-05333-5

Fig. 3 ∣. C-terminal cyclic imides are degrons that promote CRBN-dependent ubiquitination and degradation.

Fig. 3 ∣

a, The sortase system used to generate degron-tagged GFP from GFP–LPETG–His6. b, In vitro ubiquitination of GFP tagged with C-terminal cyclic imide with K0 ubiquitin. c, Quantification of ubiquitinated protein band intensity in the experiment shown in b across three replicates. d, In vitro ubiquitination of GFP tagged with uncyclized C-terminal glutamine or asparagine with K0 ubiquitin. e, Quantification of ubiquitinated protein band intensity in experiment shown in d across three replicates. f, Flow cytometry analysis of GFP in wild-type or CRBN-knockout (KO) HEK293T cells 6 h after electroporation with GFP tagged with the indicated peptide. g, Flow cytometry analysis of GFP in HEK293T cells 6 h after electroporation with GFP tagged with the indicated peptide. GFP–His6, GFP–His6 (no sortase treatment). h, Flow cytometry analysis of the GFP in HEK293T cells 6 h after electroporation with GFP tagged with the indicated peptide, with or without lenalidomide competition (100 μM). Data are mean ± s.d. One-way ANOVA with Šídák’s multiple comparisons test. P-values are shown. Western blot and flow cytometry data are representative of three independent replicates. For uncropped western blot images, see Supplementary Fig. 7.